1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. DESCRIPTION OF THE FIGS.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Methods for Cloning and Subcloning by Homologous Recombination
5.1.1 Approach 1: Introduction of Vector into Host Cell Containing Target DNA
5.1.2 Approach 2: Co-Introduction of Vector and Target DNA into the Host Cell
5.1.3 Approach 3: Introduction of Target DNA into Host Cells Containing Vector DNA
5.2 Compositions for Cloning and Subcloning by Homologous Recombination
5.2.1 The Homology Cloning Vector
5.2.1.1 The Origin of Replication
5.2.1.2 The Selectable Marker
5.2.1.3 the Homology Arms
5.2.1.4 Adapter Oligonucleotide Homology Arms
5.2.1.5 Construction of the Vector
5.2.2 Bacterial Recombinases
5.2.2.1 Protein Expression
5.2.3 Host Cells
5.2.4 Target DNA
5.3 Methods for use of the Invention
5.3.1 Introduction of DNA into Host Cells
5.3.2 Oligonucleotides
5.3.3 DNA Amplification
5.4 Methods for Diagnostic Applications
5.4.1 Detection of Foreign DNA
5.4.2 Diagnosis of Mutations and Polymorphisms in Cellular DNA
5.5 Kits
6. EXAMPLE: RECE/T AND REDxcex1/xcex2 SUBCLONING
6.1 Methods and Materials
6.2 Results
The present invention is directed to methods and compositions for DNA cloning and subcloning using bacterial recombinase-mediated homologous recombination. In a specific embodiment, RecE/T or Redxcex1/xcex2 recombinases, or any functionally equivalent system for initiating bacterial homologous recombination, such as erf from phage P22, are used. In particular, the invention relates to cloning methods, diagnostic methods, compositions comprising polynucleotides useful as cloning vectors, cells comprising such polynucleotide compositions, and kits useful for RecE/T and Redxcex1/xcex2 mediated cloning.
DNA cloning and subcloning in E. coli are fundamental to molecular biology. DNA cloning refers to the process whereby an origin of replication is operably linked to a double-stranded DNA fragment, and propagated in E. coli, or other suitable host. DNA subcloning refers to the process whereby a double-stranded DNA fragment is taken from a DNA molecule that has already been amplified, either in vitro, for example by PCR, or in vivo by propagation in E. coli or other suitable host, and is then linked to an operable origin of replication. Cloning and subcloning in E. coli is typically performed by ligating the ends of a DNA fragment to the ends of a linearized vector containing an E. coli origin of replication and a selectable marker. The selectable marker is included in the vector to ensure that the newly cloned product, the plasmid containing the insert, is retained and propagated when introduced into its E. coli host cell.
Conventional cloning methods have certain limitations. For example, since conventional cloning requires the use of restriction enzymes, the choice of DNA fragments is limited by the availability of restriction enzyme recognition sites in the DNA region of interest. Restriction sites must be found that cut the boundaries of, but not within, the desired DNA fragment. Since most useful restriction enzymes cut fairly frequently, the size of the linear DNA fragment made is also limited.
The increasing use of the polymerase chain reaction (PCR) for generating DNA fragments presents a second major drawback to conventional subcloning. The ends of PCR products are inefficient in ligation reactions due to non-templated nucleotides added to the 3xe2x80x2 termini of amplified PCR products by thermostabile polymerase. Furthermore, the use of PCR entails a high risk of mutations. Thus, molecular biologists have searched for new, more effective methods for cloning fragments of DNA, particularly when such fragments are longer than those conveniently accessible by restriction enzyme or PCR methodologies.
Homologous recombination is an alternative approach for cloning and subcloning DNA fragments. Methods for subcloning PCR products in E. coli that exploit the host""s homologous recombination systems have been described (Oliner et al., 1993, Nucleic Acids Res. 21:5192-97; Bubeck et al., 1993, Nucl. Acids. Res. 21:3601-3602). In such methods, PCR primers, designed to contain terminal sequences homologous to sequences located at the ends of a linearized vector, are used to amplify a DNA fragment of interest. The PCR product and the linearized vector are then introduced into E. coli. Homologous recombination within the E. coli host cell results in insertion of the PCR product sequences into the plasmid vector. Although these methods have been shown to be useful for subcloning PCR fragments, they have not been applied to subcloning long DNA fragments, or to cloning DNA fragments of any size.
Another method describes an in vivo subcloning method in which two linear DNA molecules, one of which has an origin of replication, and which have long regions of homology at their ends, are used to transform an E. coli sbcBC host cell. Homologous recombination occurs in vivo, and results in circularization and propagation of the newly formed plasmid (Degryse, 1996, Gene 170:45). Subsequently, the ability of E. coli sbcBC host cells to mediate homologous recombination has been applied to subcloning large DNA fragments from adenovirus and herpes virus genomic DNAs (Chartier et al., 1996, J. Virol. 70: 4805; Messerle, et al., 1997, Proc. Natl. Acad. Sci. USA 94, 14759-14763; He, 1998, Proc. Natl Acad. Sci. USA 95:2509-2514). As described, each subcloning by homologous recombination in E. coli sbcBC host cells requires at least two preparatory subcloning steps to position long homology regions either side of an E. coli origin of replication. Furthermore, DNA cloning in E. coli sbcBC strains has not been described.
Recently, homologous recombination, mediated by either RecE/RecT (RecE/T) or Redxcex1/Redxcex2 (Redxcex1/xcex2) has been shown to be useful for manipulating DNA molecules in E. coli (Zhang et al, 1998, Nature Genetics, 20, 123-128; Muyrers et al., 1999, Nucleic Acids Res. 27: 1555-1557). These papers show that, in E. coli, any intact, independently replicating, circular DNA molecule can be altered by RecE/T or Redxcex1/xcex2 mediated homologous recombination with a linear DNA fragment flanked by short regions of DNA sequence identical to regions present in the circular molecule. Integration of the linear DNA fragment into the circular molecule by homologous recombination replaces sequences between its flanking sequences and the corresponding sequences in the circular DNA molecule.
Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
The present invention provides methods and compositions for DNA cloning and subcloning using bacterial recombinase-mediated homologous recombination. The bacterial recombinase is preferably RecE/T and/or Redxcex1/xcex2. Methods can be used to clone, subclone, propagate, and amplify a polynucleotide or mixture of polynucleotides of interest using a vector comprising short regions of DNA homologous to sequences flanking a designated target DNA sequence of interest and an origin of replication.
In one embodiment, the invention provides a method for introducing a double-stranded target DNA into a vector comprising culturing a bacterial cell that expresses a functional recombinase, said bacterial cell containing (a) the target DNA comprising a first double-stranded terminus and a second double-stranded terminus, and (b) a vector DNA comprising, in the following order along the vector DNA strand: (i) a first double-stranded homology arm (ii) an origin of replication; and (iii) a second double-stranded homology arm, such that the sequence of a vector DNA strand of the first homology arm is homologous to the sequence of a target DNA strand of the first terminus, and the sequence of a vector DNA strand of the second homology arm is homologous to the sequence of the target DNA strand of the second terminus, such that the target DNA is inserted into the vector DNA between the homology arms.
In another embodiment, a method is provided for making a recombinant DNA molecule comprising: a) introducing a double-stranded vector into a cell, said cell containing a double-stranded target DNA and expressing a bacterial recombinase, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, one strand of the origin of replication, and a second homology arm; said target DNA comprising a target DNA sequence and two termini, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, the target DNA sequence, and a second terminus, such that the sequence of the first homology arm on said vector DNA strand is homologous to the sequence of the first terminus on said target DNA strand, and the sequence of the second homology arm on said vector DNA strand is homologous to the sequence of the second terminus on said target DNA strand; and b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In another embodiment, a method is provided for making a recombinant DNA molecule comprising: a) introducing a double-stranded vector and first and second double-stranded oligonucleotides into a cell, said cell containing a double-stranded target DNA and expressing a bacterial recombinase, said vector comprising an origin of replication and two double-stranded homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication, and a second homology arm; said target DNA comprising a target DNA sequence and two double-stranded termini, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, a target DNA sequence, and a second terminus; said first oligonucleotide comprising a first oligonucleotide DNA strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a first nucleotide sequence and a second nucleotide sequence, said first nucleotide sequence being homologous to the nucleotide sequence of the first homology arm on said vector DNA strand, and said second nucleotide sequence being homologous to the nucleotide sequence of the first terminus on said target DNA strand; said second oligonucleotide comprising a second oligonucleotide strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2, a third nucleotide sequence and a fourth nucleotide sequence, said third nucleotide sequence being homologous to the nucleotide sequence of the second homology arm on said vector DNA strand and said fourth nucleotide sequence being homologous to the nucleotide sequence of the second terminus on said target DNA strand; and b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In another embodiment, a method is provided for making a recombinant DNA molecule comprising: a) introducing a double-stranded target DNA molecule into a cell, said cell containing a vector and expressing a bacterial recombinase, said target DNA comprising a target DNA sequence and two double-stranded termini, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, a target DNA sequence, and a second terminus; said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; such that the sequence of the first homology arm on said vector DNA strand is homologous to the sequence of the first terminus on said target DNA strand, and the sequence of the second homology arm on said vector DNA strand is homologous to the sequence of the second terminus on said target DNA strand; and b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In another embodiment, a method is provided for making a recombinant DNA molecule comprising: a) introducing a double-stranded target DNA molecule and a first and second double-stranded oligonucleotide into a cell, said cell containing a vector and expressing a bacterial recombinase, said target DNA comprising a target DNA sequence and two termin, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, a target DNA sequence, and a second terminus; said first oligonucleotide comprising a first oligonucleotide DNA strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a first nucleotide sequence and a second nucleotide sequence, said first nucleotide sequence being homologous to the nucleotide sequence of the first homology arm on said vector DNA strand, and said second nucleotide sequence being homologous to the nucleotide sequence of the first terminus on said target DNA strand; said second oligonucleotide comprising a second oligonucleotide strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2, a third nucleotide sequence and a fourth nucleotide sequence, said third nucleotide sequence being homologous to the nucleotide sequence of the second homology arm on said vector DNA strand and said fourth nucleotide sequence being homologous to the nucleotide sequence of the second terminus on said target DNA strand; and said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; and b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In another embodiment, a method is provided for making a recombinant DNA molecule comprising: a) introducing a double-stranded vector and a double-stranded target DNA into a cell expressing a bacterial recombinase, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm, said target DNA comprising a target DNA sequence and two termini, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, a target DNA sequence; and a second terminus; such that the nucleotide sequence of the first homology arm on said vector DNA strand is homologous to the nucleotide sequence of the first terminus on said target DNA strand, and the nucleotide sequence of the second homology arm on said vector DNA strand is homologous to the sequence of the second terminus on said target DNA strand; and
b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In a specific embodiment, of this method the host cell further contains a nucleotide sequence encoding a site-specific recombinase operatively linked to a promoter, and the vector further comprises a first and second recognition site for the site-specific recombinase, a first recognition site located outside the first and second homology arms, and a second site-specific recombinase recognition site located inside the first and second homology arms; and during or after step b), inducing expression of the site-specific recombinase.
In another specific embodiment of this method, the host cell further contains a nucleotide sequence encoding a site-specific endonuclease operatively linked to a promoter, and the vector further comprises a recognition site for the site-specific endonuclease located inside the first and second homology arms; and during or after step b), inducing expression of the site-specific endonuclease.
In another embodiment, the inventions provides a method for making a recombinant DNA molecule comprising: a) introducing a double-stranded vector, a double-stranded target DNA molecule, and a first and second double-stranded oligonucleotide into a cell expressing a bacterial recombinase, said vector comprising an origin of replication and two double-stranded homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; said target DNA comprising target DNA sequence and two double-stranded termini, in the following order, from 3xe2x80x2 to 5xe2x80x2 along a target DNA strand: a first terminus, a target DNA sequence, and a second terminus; said first oligonucleotide comprising a first oligonucleotide DNA strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a first nucleotide sequence and a second nucleotide sequence, said first nucleotide sequence being homologous to the nucleotide sequence of the first homology arm on said vector DNA strand, and said second nucleotide sequence being homologous to the sequence of the first terminus on said target DNA strand; said second oligonucleotide comprising a second oligonucleotide strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2, a third nucleotide sequence and a fourth nucleotide sequence, said third nucleotide sequence being homologous to the nucleotide sequence of the second homology arm on said vector DNA strand and said fourth nucleotide sequence being homologous to the nucleotide sequence of the second terminus on said target DNA strand; and b) subjecting the cell to conditions that allow intracellular homologous recombination to occur.
In a specific embodiment of this method, the host cell further contains a nucleotide sequence encoding a site-specific recombinase operatively linked to a promoter, and the vector further comprises a first and second recognition site for the site-specific recombinase, a first recognition site located outside the first and second homology arms, and a second site-specific recombinase recognition site located inside the first and second homology arms; and during or after step b), inducing expression of the site-specific recombinase.
In another specific embodiment of this method, wherein the host cell further contains a nucleotide sequence encoding a site-specific endonuclease operatively linked to a promoter, and the vector further comprises a recognition site for the site-specific endonuclease located inside the first and second homology arms; and during or after step b), inducing expression of the site-specific endonuclease.
In specific embodiments, the vector further comprises a selectable marker located outside the homology arms, such that the vector comprises, in either of the following two orders from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: i) the first homology arm, the selectable marker, the origin of replication and the second homology arm, or ii) the first homology arm, the origin of replication, the selectable marker, and the second homology arm. In a specific embodiment, the selectable marker confers antibiotic resistance to the cell containing the vector.
In various specific embodiments, the bacterial recombinase is RecE/T or Redxcex1/xcex2 recombinase or both RecE/T and Redxcex1/xcex2. In other specific embodiments, the cell is a bacterial cell. In other specific embodiments, the cell is an E. coli cell. In other specific embodiments, the cell eukaryotic cell that recombinantly expresses RecE/T and/or Redxcex1/xcex2 protein. In other specific embodiments, the method further comprises isolating a recombinant DNA molecule that comprises the target DNA inserted into the vector.
In another embodiment, the invention provides a double-stranded DNA vector useful for directed cloning or subcloning of a target DNA molecule of interest, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; such that the nucleotide sequence of the first homology arm on a first vector DNA strand is homologous to the sequence of the first terminus on a first target DNA strand, and the nucleotide sequence of the second homology arm on the first vector DNA strand is homologous to the nucleotide sequence of the second terminus on the first target DNA strand. In a specific embodiment of the vector, the origin of replication is a bacterial origin of replication. In another specific embodiment, the origin of replication functions in E. coli. In another specific embodiment, the origin of replication functions in a mammalian cell.
The invention further provides a cell comprising a double-stranded DNA vector useful for directed cloning or subcloning of a target DNA molecule of interest, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; such that the nucleotide sequence of the first homology arm on a first vector DNA strand is homologous to the sequence of the first terminus on a first target DNA strand, and the nucleotide sequence of the second homology arm on the first vector DNA strand is homologous to the nucleotide sequence of the second terminus on the first target DNA strand. In a specific embodiment, the cell is a bacterial cell.
The invention further provides a kit useful for directed cloning or subcloning of a target DNA molecule comprising in one or more containers: a) a double-stranded DNA vector useful for directed cloning or subcloning of a target DNA molecule of interest, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; such that the nucleotide sequence of the first homology arm on a first vector DNA strand is homologous to the sequence of the first terminus on a first target DNA strand, and the nucleotide sequence of the second homology arm on the first vector DNA strand is homologous to the nucleotide sequence of the second terminus on the first target DNA strand; and b) a cell containing a bacterial recombinase. In a specific embodiment of the kit, the homology arms have sequence homology to a BAC, PAC, lambda, plasmid or YAC based cloning vector. In another specific embodiment of the kit, the first and second double-stranded oligonucleotide have nucleotide sequence homology to a BAC, PAC, lambda, plasmid or YAC based cloning vector.
In another embodiment, a kit useful for directed cloning or subcloning of a target DNA molecule is provided comprising in one or more containers: a) a double-stranded DNA vector useful for directed cloning and subcloning of a target DNA molecule of interest, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; b) a first double-stranded oligonucleotide comprising a first oligonucleotide DNA strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a first sequence and a second sequence, said first nucleotide sequence being homologous to the nucleotide sequence of the first homology arm on said vector DNA strand, and said second nucleotide sequence being homologous to the nucleotide sequence of a first terminus on a target DNA strand; c) a second double-stranded oligonucleotide comprising a second oligonucleotide strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a third nucleotide sequence and a fourth nucleotide sequence, said third nucleotide sequence being homologous to the nucleotide sequence of the second homology arm on said vector DNA strand and said fourth nucleotide sequence being homologous to the nucleotide sequence of a second terminus on said target DNA strand; and d) a cell containing a bacterial recombinase. In a specific embodiment of the kit, the cell is an E. coli cell. In another specific embodiment of the kit, the cell is a frozen cell competent for uptake of DNA.
In another embodiment, the invention provides a kit useful for directed cloning or subcloning of a target DNA molecule comprising in one or more containers: a) a double-stranded DNA vector useful for directed cloning and subcloning of a target DNA molecule of interest, said vector comprising an origin of replication and two homology arms, in the following order from 5xe2x80x2 to 3xe2x80x2 along a vector DNA strand: a first homology arm, the origin of replication and a second homology arm; b) a first double-stranded oligonucleotide comprising a first oligonucleotide DNA strand comprising, in the following order, from 3xe2x80x2 to 5xe2x80x2: a first nucleotide sequence and a second nucleotide sequence, said first nucleotide sequence being homologous to the nucleotide sequence of the first homology arm on said vector DNA strand, and said second nucleotide sequence being homologous to the nucleotide sequence of a first terminus on a target DNA strand; and c) a second double-stranded oligonucleotide comprising a second oligonucleotide strand comprising, in the following order, from 3xe2x80x2 to 5: a third nucleotide sequence and a fourth nucleotide sequence, said third nucleotide sequence being homologous to the nucleotide sequence of the second homology arm on said vector DNA strand and said fourth sequence being homologous to the nucleotide sequence of a second terminus on said target DNA strand. In a specific embodiment of the kit, the DNA vector is purified. In another embodiment of the kit, the DNA vector, the first double-stranded oligonucleotide, and the second double-stranded oligonucleotide are purified.
In other specific embodiments of kits provided by the invention the target DNA molecule comprises bacterial, viral, parasite, or protozoan DNA. In other specific embodiments, the target DNA molecule comprises a genetic mutation or polymorphism known or suspected to be associated with a disorder or disease. In other specific embodiments, the bacterial recombinase is RecE/T or Redxcex1/xcex2 recombinase or both RecE/T and Redxcex1/xcex2 recombinases.
The methods of the invention may be used in diagnostics. For example, plasmids or linear DNA fragments may be designed to capture a specific DNA target to detect its presence in a sample from a subject e.g., a viral DNA present in a patient""s sample. In one embodiment, the invention provides methods for detection of target DNA known or suspected to be associated with a disorder or disease when genetically mutated. In specific embodiments, the target DNA is a bacterial, viral, parasite, or protozoan DNA. In a specific embodiment, a method is provided which further comprise detecting a recombinant DNA molecule that comprises the target DNA inserted into the vector. In another embodiment, the method further comprises detecting a recombinant DNA molecule that comprises the target DNA inserted into the vector.
In another embodiment, the invention provides a method of detecting the presence of an infectious agent wherein the target DNA is derived from a patient suspected of having the infectious disease, and the sequences of the first and second homology arms are homologous to the sequences present in DNA of the infectious agent. In a specific embodiment, the target DNA is derived from a patient suspected of having the infectious disease, and said second and fourth nucleotide sequences are homologous to sequences present in DNA of the infectious agent. In other specific embodiments, the infectious agent is a virus, bacteria, protozoa, fungus, or parasite.
In another embodiment, a method is provided for detecting the presence of a genetic condition, disease, disorder, or polymorphic trait, wherein the target DNA is derived from a patient suspected of having a genetic condition, disease, disorder, or polymorphic trait, and the sequence of the first homology arm is homologous to the sequence upstream from a site known or suspected to be associated with the genetic condition, disease, disorder, or polymorphic trait, and the sequence of the second homology arm is homologous to the sequence downstream from a site known or suspected to be associated with the genetic condition, disease, disorder, or polymorphic trait. In a specific embodiment, a method is provided for detecting the presence of a genetic condition, genetic disease, genetic disorder, or polymorphic trait wherein the target DNA is derived from a patient suspected of having the genetic condition, genetic disease, genetic disorder, or polymorphic trait, and the sequence of the first double-stranded oligonucleotide is homologous to the sequence upstream from a site known or suspected to be associated with the genetic condition, genetic disease, genetic disorder, or polymorphic trait, and the sequence of the second double-stranded oligonucleotide is homologous to the sequence downstream from a site known or suspected to be associated with the genetic condition, genetic disease, genetic disorder, or polymorphic trait. In a specific embodiment, the genetic condition, genetic disease, genetic disorder, or polymorphic trait is or predisposes the patient to cancer, asthma, arthritis, drug resistance, drug toxicity, or a neural, neuropsychiatric, metabolic, muscular, cardiovascular, or skin condition, disease or disorder.